Draft genome sequence and intact prophage characterisation of multidrug-resistant Acinetobacter baumannii isolate ABU3 from a tracheal suction specimen in Thailand

Abstract

Multidrug-resistant Acinetobacter baumannii ABU3 was isolated from a tracheal suction specimen from a 76-year-old female patient in Thailand. The draft genome was sequenced using Illumina NextSeq 550. The genome comprised 95 contigs totaling 3826,273 base pairs with an N50 of 112,717 bp and GC content of 38.89. Digital DNA-DNA hybridisation with A. baumannii ATCC 19606^T revealed 80.3 % similarity. MLST assigned the isolate to ST-164, whereas MLST of the core genome showed 99.0 % similarity to cgST-4302. Resistance gene analysis identified bla_OXA-23, bla_CARB variants, bla_ADC-25, and tet(39). Antimicrobial susceptibility testing showed resistance to carbapenems, fluoroquinolones, beta-lactam combinations, extended-spectrum cephalosporins, and penicillins, with sensitivity to aminoglycosides and folate pathway inhibitors. Two intact prophage regions (23.5 and 26.3 kb) were identified containing structural and functional components. Data are available at NCBI under BioProject PRJNA550309.

Specifications Table

 

Subject	Biological sciences
Specific subject area	Omics: Genomics
Data format	Raw and analysed
Type of data	Tables, figures
Data collection	Genomic DNA was extracted using PureLink™ Genomic DNA Mini Kit and sequenced on the NextSeq 550 platform (2 × 150-bp reads). Quality control and adapter trimming were performed using fastp v0.23.4. De novo genome assembly was performed using Unicycler v0.5.0, with quality assessment using QUAST v5.0.2 and CheckM2 v1.0.1. Taxonomic identification and phylogenomic analysis used TYGS. MLST analysis used the PubMLST database. Genome annotation using NCBI PGAP. Antimicrobial resistance genes were identified using ResFinder v4.4.2. Prophage regions were detected using PHASTEST.
Data source location	A. baumannii ABU3 was isolated from a tracheal suction specimen from a 76-year-old female patient in the surgical ward at Srinagarind Hospital, Khon Kaen province, Thailand (16°28′5.45″N, 102°49′48.21″E) in September 2018.
Data accessibility	Sequencing data were deposited in the National Center for Biotechnology Information (NCBI) Genbank database under accession number JBPRPP000000000. The deposited draft genome sequencing data are available at https://www.ncbi.nlm.nih.gov/nuccore/JBPRPP000000000.

1. Value of the Data

• •The draft genome data aids comparative genomic studies of multidrug-resistant Acinetobacter species.
• •The draft genome data facilitates A. baumannii antimicrobial resistance gene identification and drug resistance phenotype prediction.
• •The intact prophage regions (23.5 and 26.3 kb) provide resources for A. baumannii bacteriophage-based therapeutic development.

2. Background

Acinetobacter baumannii has become clinically important because of its extensive antibiotic resistance, which frequently results in limited treatment options [1]. The World Health Organisation (WHO) designated A. baumannii as a priority pathogen, highlighting the urgent need for innovative therapeutic approaches [2]. Bacteriophage-based interventions offer several compelling advantages against this multidrug-resistant pathogen, including efficiency and highly specific targeting of A. baumannii while preserving human cells and beneficial microbiota. Phages can be discovered from multiple sources, including natural environmental samples such as water and soil [3], clinical specimens from infected patients [4], and bacterial genomes as integrated prophage elements [5]. Identifying phages within bacterial genomes provides a more targeted approach, offering stable and genetically integrated prophage sources that can be analysed and potentially reactivated for therapeutic applications against A. baumannii. Genomic analysis has become a key strategy for identifying prophage regions within bacterial genomes [6]. This approach enables direct bacteriophage detection without isolation requirements while maintaining high selectivity, as current sequencing technologies can effectively identify minimal phage sequences in bacterial genomes or environmental samples. This study investigated the genomic structure of A. baumannii to locate prophage sequences for developing targeted therapeutic applications against this priority pathogen.

3. Data Description

This study reports the draft genome sequence data of A. baumannii ABU3, with the circular genome representation depicting the overall genomic architecture (Fig. 1).

Fig. 1.

Circular genome representation of A. baumannii ABU3 draft assembly (95 contigs, 3826,273 bp total) constructed using Proksee software [7]. Blue bars denote protein-coding sequences (CDSs), whilst grey arrows indicate individual contigs. The green and purple peaks represent positive and negative GC skew values, respectively, and the black peaks show the GC content distribution.

The genome is composed of 95 contigs with a total size of 3826,273 bp, an N50 value of 112,717 bp, and a GC content of 38.89 % (Table 1).

Table 1.

Genomic features and assembly statistics of ABU3.

Attribute	A. baumannii ABU3
Genome size (bp)	3826,273
Number of contigs	95
Genome coverage	519×
GC content ( %)	38.89
Largest contig (bp)	265,436
N50	112,717
N90	33,508
L50	12
L90	35
Total gene	3716
Total CDS	3645
tRNA	64
rRNA	3
ncRNA	4

The ABU3 draft genome demonstrated 100 % completeness with 0.1 % detected contamination. Digital DNA-DNA hybridisation (dDDH) analysis with A. baumannii ATCC 19606^T yielded 80.3 % similarity, confirming species-level identification. Phylogenomic analysis further validated ABU3 as A. baumannii (Fig. 2). Multilocus sequence typing (MLST) analysis revealed contrasting results between schemes: the Oxford scheme showed 85.7 % similarity to ST-176, whereas the Pasteur scheme definitively assigned the isolate to ST-164. Core genome analysis demonstrated 99.0 % similarity to cgST-4302. The moderate Oxford similarity likely reflects sequence variations from draft genome assembly limitations rather than true genetic divergence. The definitive ST-164 assignment provides robust epidemiological classification, while the high cgMLST similarity indicates close evolutionary relationships with characterised isolates, offering valuable epidemiological insights. These contrasting results highlight the importance of MLST approaches for comprehensive A. baumannii characterisation, given the species’ genetic complexity. Antimicrobial susceptibility testing (AST) based on whole-genome sequencing (WGS) revealed the presence of several known multidrug resistance genes in the ABU3 genome of A. baumannii. These genes include bla_ADC-25 (95.01–96.11 %), bla_CARB-5 (99.89 %), bla_CARB-16 (99.89 %), bla_CARB-49 (99.89 %), bla_OXA-23 (99.88 %), bla_OXA-91 (100.00 %), and tet(39) (100.00 %) (Table 2).

Fig. 2.

Phylogenomic reconstruction based on the draft genome sequences of A. baumannii ABU3 and related type strains via the TYGS platform. Branch values represent pseudo-bootstrap support exceeding 60 % from 100 iterations using Genome Blast Distance Phylogeny (GBDP), with mean branch support of 88.2 %.

Table 2.

Identification of antimicrobial resistance genes and prediction of resistance phenotypes based on the ABU3 genome sequence.

Resistance gene	Identity (%)	Alignment length/gene length	Position in the reference	Contig	Position in the contig	Resistance phenotype	Antibiotic class	GenBank accession no.
blaADC-25	96.11	849/1152	1–848	22	58,887–59,734	Unknown Beta-lactam	Beta-lactam	EF016355
blaADC-25	95.01	892/1152	1–846	86	8–869	Unknown Beta-lactam		EF016355
blaCARB-16	99.89	897/897	1–897	74	710–1606	Ampicillin, Amoxicillin, Piperacillin		HF953351
blaCARB-49	99.89	897/897	1–897	74	710–1606	Ampicillin, Amoxicillin, Piperacillin		KX599396
blaCARB-5	99.89	897/897	1–897	74	710–1606	Ampicillin, Amoxicillin, Piperacillin		AF135373
blaOXA-23	99.88	822/822	1–822	70	1590–2411	Imipenem, Meropenem		AY795964
blaOXA-91	100.00	825/825	1–825	17	21,863–22,687	Unknown Beta-lactam		DQ519086
tet(39)	100.00	1122/1122	1122	73	872–1993	Doxycycline, Tetracycline	Tetracycline	KT346360

Resistance gene analysis identified bla_ADC-25, bla_CARB variants, bla_OXA-23, bla_OXA-91, and tet(39) conferring resistance to beta-lactam and tetracycline classes (Table 2).

Antimicrobial susceptibility testing confirmed the multidrug-resistant (MDR) phenotype, with resistance observed across five antibiotic classes: carbapenems, fluoroquinolones, beta-lactam combinations, extended-spectrum cephalosporins, and penicillins, while the isolate remained sensitive to aminoglycosides and folate pathway inhibitors (Table 3). The draft genome sequence data facilitate comprehensive analysis of A. baumannii antimicrobial resistance and prophage detection.

Table 3.

Antimicrobial susceptibility profile of A. baumannii ABU3 against seventeen antimicrobial agents.

Antimicrobial category	Antimicrobial agent	Susceptibility result
Aminoglycosides	Amikacin	Susceptible
	Gentamicin	Susceptible
	Netilmicin	Susceptible
Antipseudomonal carbapenems	Imipenem	Resistant
	Meropenem	Resistant
	Ertapenem	Resistant
Antipseudomonal fluoroquinolones	Levofloxacin	Resistant
Antipseudomonal penicillins + ß-lactamase inhibitors	Piperacillin-tazobactam	Resistant
Extended-spectrum cephalosporins	Cefotaxime	Resistant
	Ceftriaxone	Resistant
	Ceftazidime	Resistant
	Cefoxitin	Resistant
	Cefuroxime	Resistant
	Cefoperazone	Resistant
Folate pathway inhibitors	Trimethoprim-sulphamethoxazole	Susceptible
Penicillins + ß-lactamase inhibitors	Augmentin	Resistant
Penicillins	Ampicillin	Resistant

Two intact prophage regions were identified within the A. baumannii ABU3 genome, designated Region 1 (23.5 kb) and Region 2 (26.3 kb), both containing substantial genetic content characteristic of complete bacteriophages (Fig. 3). Region 1 (Fig. 3) received a PHASTEST score of 150 and was on contig 36 (genomic positions 7293–30,860 bp). This region encodes 22 predicted proteins, including 21 phage-related proteins and one hypothetical protein. Comparative analysis showed the highest similarity to Pseudomonas phage phiCTX, suggesting cross-genus phage relationships that may indicate horizontal gene transfer between bacterial hosts. Region 2 (Fig. 3) achieved a PHASTEST score of 100 and was positioned on contig 39 (genomic positions 337–26,729 bp). This region contains 21 phage-related genes. The region exhibited the greatest similarity to the Acinetobacter phage Bphi_B1251, indicating host-specific phage evolution within the Acinetobacter genus. Both prophage regions exhibited well-organised gene clusters that responded to bacteriophage functions. Structural genes are clustered in dedicated regions for DNA packaging (terminase and portal proteins), head formation (head proteins), and tail assembly (tail proteins, fibre proteins, and plate proteins). All essential structural components were detected in both prophage regions. These features included complete gene clusters for phage structural proteins.

Fig. 3.

Intact prophage regions in A. baumannii ABU3 Prophage regions 1 (23.5 kb) and 2 (26.3 kb) identified by PHASTEST analysis. Genes are colour-coded by the predicted function, as indicated in the legend. Both regions contain essential phage structural and functional components arranged in organised gene clusters typical of complete bacteriophages.

4. Experimental Design, Materials, and Methods

4.1. Bacterial isolation

In September 2018, a clinical isolate of A. baumannii (isolate ABU3) was obtained from a tracheal suction specimen from a 76-year-old female patient in the surgical ward at Srinagarind Hospital, Khon Kaen province, Thailand (16°28′5.45″N, 102°49′48.21″E). The specimens were cultured on Blood agar and MacConkey agar plates and incubated overnight at 37 °C. A single colony was identified using an automated microbial identification system VITEK^Ⓡ2 COMPACT (bioMérieux, France). ABU3 was selected and subcultured on Nutrient agar (NA). Subsequently, the isolate was grown in Nutrient broth (NB) at 37 °C with shaking at 150 rpm for 18 h and preserved in NB containing 30 % v/v glycerol at −80 °C.

4.2. Preparation of genomic DNA

Genomic DNA was extracted from overnight ABU3 cultures using the PureLink™ Genomic DNA Mini Kit (Invitrogen, USA) according to the manufacturer’s protocol. DNA concentration and quality were subsequently assessed using NanoDrop spectrophotometry (Thermo Scientific, USA) and agarose gel electrophoresis.

4.3. Whole-genome sequencing and assembly

Sequencing libraries were prepared from 1 ng of DNA using the Nextera XT DNA library preparation kit (Illumina, CA, USA). Raw sequencing reads were generated using a NextSeq 550 sequencer (Illumina, CA, USA) with a NextSeq 500/550 high output kit v2.5 (300 cycles, 2 × 150-bp reads). The raw reads were subjected to quality assessment, adapter trimming, and quality filtering using fastp v0.23.4 with default parameters [8]. De novo genome assembly was subsequently performed using Unicycler v0.5.0 with standard settings [9], and the assembly quality was evaluated using QUAST v5.0.2 with default parameters [10].

4.4. Taxonomic identification and molecular typing

We evaluated the quality of the genome sequence using CheckM2 v1.0.1 with default parameters [11]. Digital DNA-DNA hybridisation (dDDH) examination and phylogenomic tree generation were conducted using draft genome sequences of ABU3 isolate and corresponding type strains via the Type (Strain) Genome Server (TYGS) [12]. Multilocus sequence typing (MLST) characterisation was performed using both the Oxford and Pasteur methodologies available through the PubMLST database [13]. Sequence types (STs) were established by retrieving allele sequences from the assembled genome and aligning them against reference alleles within the database. Core genome MLST (cgMLST) examination was implemented using the cgMLST v1 methodology to achieve enhanced resolution in molecular characterisation [13]. The most closely related profiles were identified through percentage similarity assessments against the existing sequence types in the database.

4.5. Genome annotation and antimicrobial gene prediction

A genomic map of isolate ABU3 was constructed using Proksee [7], and genome annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) with default settings [14]. The draft genome sequence data was analysed for antimicrobial resistance genes using ResFinder v4.4.2 with standard parameters [15].

4.6. Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was conducted using the automated Sensititre™ ARIS™ 2X system with Sensititre Gram Negative MDRGN2F AST plates (Thermo Fisher Scientific, USA). The ABU3 strain was tested against seventeen antimicrobial agents representing eight major categories including aminoglycosides, antipseudomonal carbapenems, antipseudomonal fluoroquinolones, antipseudomonal penicillins with β-lactamase inhibitors, extended-spectrum cephalosporins, folate pathway inhibitors, penicillins with β-lactamase inhibitors, and penicillins. Interpretation of the results was conducted according to Clinical and Laboratory Standards Institute (CLSI) guidelines [16], and multidrug resistance (MDR) classification followed the international consensus criteria established by Magiorakos et al. (2012) [17].

4.7. Prophage genome detection and analysis

Prophage regions within the ABU3 genome were identified using PHASTEST with default parameters [18]. Only intact prophage regions (score >90), representing complete and potentially functional prophages, were analysed based on their completeness and functional potential.

Limitations

Although next-generation sequencing produces substantial volumes of data, the resulting de novo genome assemblies frequently exhibit incomplete coverage. Such assemblies may have limitations that render them susceptible to annotation inaccuracies, particularly regarding imprecise gene prediction within draft genomes such as ABU3.

Ethics Statement

This study was approved by the Human Research Ethics Committee of Thammasat University (Science) (HREC-TUSc) under project number 68DR074 (COE No 018/2568).

Data Availability

• INSDCAcinetobacter baumannii strain ABU3, whole genome shotgun sequencing project. (Original data).